Dosage regimen for administering a CD19XCD3 bispecific antibody

ABSTRACT

The disclosure provides a method for assessing the risk of potential adverse effects for a human patient mediated by the administration of a CD19×CD3 bispecific antibody to said patient comprising determining the ratio of B cells to T cells of said patient. The disclosure also provides a method for administering a CD19×CD3 bispecific antibody to a human patient having a B:T cell ratio of about 1:5 or lower, comprising administering doses in a dosing regimen. This dosing regimen can be applied in methods for treating malignant CD19 positive lymphocytes or for ameliorating and/or preventing an adverse effect mediated by the administration of said bispecific antibody. The Also provided is a pharmaceutical package or kit comprising a first dose and a second dose and optionally a third dose of said antibody.

The present invention relates to a method for assessing (analyzing) therisk of potential adverse effects for a human patient mediated by theadministration of a CD19×CD3 bispecific antibody to said patientcomprising determining the ratio of B cells to T cells of said patient,wherein a ratio of about 1:5 or lower is indicative for a risk ofpotential adverse effects for said patient. Accordingly, the presentinvention relates a method (dosage regimen) for administering a CD19×CD3bispecific antibody to a human patient having a B:T cell ratio of about1:5 or lower, comprising (a) administering a first dose of said antibodyfor a first period of time; and consecutively (b) administering a seconddose of said antibody for a second period of time, wherein said seconddose exceeds said first dose. In some embodiments, a third dose of saidantibody is administered for a third period of time. This dosage regimencan be applied in methods for treating malignant CD19 positivelymphocytes or for ameliorating and/or preventing an adverse effectmediated by the administration of said bispecific antibody. The presentinvention also relates to the use of a CD19×CD3 bispecific antibody forthe preparation of a pharmaceutical composition to be used in a methodof the present invention. A pharmaceutical package or kit comprising afirst dose and a second dose and optionally a third dose of saidantibody as defined in the methods/dosage regimen of the presentinvention is disclosed as well.

Antibody-based cancer therapies require a target antigen firmly bound tothe surface of cancer cells in order to be active. By binding to thesurface target, the antibody can directly deliver a deadly signal to thecancer cell or indirectly by, for example, recruiting a cytotoxic Tcell, if it is a bispecific antibody. In an ideal treatment scenario, atarget antigen is abundantly present and accessible on every cancer celland is absent, shielded or much less abundant on normal cells. Thissituation provides the basis for a therapeutic window in which a definedamount of the antibody-based therapeutic effectively hits cancer cellsbut spares normal cells.

Though antibodies are an effective means in treating many disorders, inparticular cancer, their administration is not necessarily devoid ofside effects. Adverse effects may cause a reversible or irreversiblechange in the health status of a patient. As adverse effects could beharmful and undesired, it is highly desirable to avoid them. However,though it is known that a medicament can cause adverse effects, itsprescription and administration could not be avoided or is accepted,since the medicament has an outstanding beneficial therapeutic effect ormay even be life-saving.

In clinical trials, a general distinction can be made between adverseeffects (AEs) and serious adverse effects (SAEs). Specifically, adverseeffects can be classified in 5 grades in accordance with the CommonTerminology Criteria for Adverse Events (CTCAE). Grade 1 relates to mildAE, Grade 2 to moderate AE, Grade 3 to severe AE, Grade 4 tolife-threatening or disabling AE, while Grade 5 means death related toAE.

An adverse effect observed in antibody therapy is the occurrence ofinfusion-related side effects, such as the cytokine release syndrome(“CRS”). Other adverse side effects described to be associated with CRSare fatigue, vomiting, tachycardia, hypertension, back pain, but alsocentral nervous system reactions (CNS reactions), such as seizures,encephalopathy, cerebral edema, aseptic meningitis, and headache.

Cytokine release and neurological reactions have not only been observedwith monoclonal antibodies binding to the T cell receptor but also witha CD19×CD3 bispecific single chain antibody binding to the CD3 part ofthe T cell receptor (called Blinatumomab (MT103)).

Blinatumomab (MT103) is a lymphoma-directed, recombinant bispecificsingle-chain CD19×CD3 antibody that binds to CD19 on the surface ofalmost all B cells and B tumor cells and concomitantly can engage a Tcell, thereby triggering the T-cell to kill the target B cell or B tumorcell. Blinatumomab consists of four immunoglobulin variable domainsassembled into a single polypeptide chain. Two of the variable domainsform the binding site for CD19, a cell surface antigen expressed on mostB cells and B tumor cells. The other two variable domains form thebinding site for the CD3 complex on T cells. Blinatumomab is designed todirect the body's cytotoxic, or cell-destroying, T cells against tumorcells, and represent a new therapeutic approach to cancer therapy.Blinatumomab is presently in clinical trials.

As described for instance in WO 99/54440, adverse effects have beenobserved in a previous study performed with Blinatumomab applied inrepeated bolus infusions to a patient with B-cell derived chroniclymphatic leukaemia (B-CLL). As shown in FIGS. 19 and 20 of WO 99/54440,release of TNF, IL-6 and IL-8 has been found in response to each of thetwo administered 20 minute-infusions of 3 microgram and 10 microgram ofthe mentioned bispecific single chain antibody, respectively, withcytokine release after each administration. Maximal cytokine release wasobserved after administration of 10 microgram of bispecific single chainantibody. In a following clinical trial study, in which escalating dosesof the CD19×CD3 bispecific single chain antibody have been administeredto patients with B cell malignancies as bolus infusions, adverse effectshave also been observed. According to a retrospective analysis, 7 out of22 patients showed an early neurological reaction, including, forexample, confusion, ataxia, speech disorder, or disorientation.

In order to try to better manage these undesired side effects, the modeof administration of the CD19×CD3 bispecific single chain antibody hasbeen changed in that it has been switched over from bolus infusion to acontinuous intravenous administration of said antibody for a longerperiod of time. As shown in Bargou et al. (Science 321 (2008): 974-7),doses as low as 0.005 milligrams per square meter per day continuouslyadministered to non-Hodgkin's lymphoma patients over four weeks led toan elimination of lymphoma target cells in blood. Partial and completetumor regressions were first observed at a dose level of 0.015milligrams/m²/d, and all seven patients treated at a dose level of 0.06milligrams/m²/d experienced a tumor regression (Bargou et al., citedabove). The CD19×CD3 bispecific single chain antibody also led toclearance of tumor cells from bone marrow and liver. However, thoughthis (still ongoing) study established clinical proof of concept for thetherapeutic potency of the CD19×CD3 bispecific single chain antibodyformat in the treatment of blood-cell derived cancer, neurologicalreactions have been found in the course of the aforementioned clinicaltrial. Accordingly, since Blinatumomab is a very promising candidatemedicament for treating non-Hodgkin's lymphoma (NHL), acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and/ormantle cell lymphoma, it is highly desirable to reduce or evencompletely avoid undesired side-effects in the treatment of patients inneed thereof with the CD19×CD3 bispecific single chain antibody.

Evidently, it is difficult to design a CD19×CD3 antibody-based therapy,which does not cause CNS (neurological) reactions including neurologicalreactions, or, to put it differently, it is desired to provide aCD19×CD13 antibody-based medical therapies with increased patienttolerability, i.e., reduced or even no undesired adverse effects such asCNS reactions.

Though pharmaceutical means and methods which allow a more gradualactivation of T cell populations (see WO 2007/068354) already helped toavoid significant adverse side effects in patients treated with theCD19×CD3 bispecific single chain antibody, neurological reactions couldunfortunately not be prevented by these measures, in particular in casesin which doses of more than 5 to 10 microgram per square meter per day(i.e. 24 h) of the antibody have been administered.

Thus, the technical problem underlying the present invention was toprovide dosage regimens and methods to overcome the above problem.

The present invention addresses this need and thus provides embodimentsconcerning methods as well as dosage regimens for administering aCD19×CD3 bispecific antibody to a human patient.

These embodiments are characterized and described herein and reflectedin the claims.

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

All publications and patents cited in this disclosure are incorporatedby reference in their entirety. To the extent the material incorporatedby reference contradicts or is inconsistent with this specification, thespecification will supersede any such material.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

In view of the adverse events, particularly the CNS events includingneurological reactions observed with antibodies, also including theCD19×CD3 bispecific antibody, the finding that the CD19×CD3 bispecificsingle chain antibody can be administered so that it is tolerated by thepatients, if it is administered in accordance with the dosage regimen asprovided herein, is definitely remarkable.

Specifically, the present inventors observed that those patients, towhom a CD19×CD3 bispecific antibody was administered, encountered CNSevents, if they had a B:T cell ratio of about 1:5 or lower. Accordingly,the present invention for the first time establishes a low B:T cellratio as a potential high risk factor for the occurrence of adverseeffects including neurological reactions in the treatment of malignantCD19 positive lymphocytes occurring in leukemias and lymphomas (seeExamples 2, 3 and 4).

Particularly, the inventors of the present application observed thatnon-Hodgkin lymphoma (NHL) patients and acute lymphoblastic leukemia(ALL) patients with a low B:T cell ratio in peripheral blood have anincreased risk for the development of an early neurological reaction.This neurological reaction occurs mainly during the first day(s) oftreatment with a CD19×CD13 bispecific antibody. In particular, themajority of the neurological reactions occurred after about 12 to 120hours after start of treatment. These neurological reactions weretransient, fully reversible and resolved without sequelae within 3 to 72hours after stop of the treatment.

The inventors made these unexpected observations in various clinicaltrial studies using the CD19×CD3 bispecific antibody:

Looking at “short-term” (bolus) infusion trials, 7 out of 22 patientshad an early neurological reaction. 6 of these 7 patients had a low B:Tcell ratio, i.e., a B:T cell ratio of about 1:5 or lower, beforetreatment. Of the remaining 15 patients without neurological reaction,only 1 patient had a low B:T cell ratio.

In an NHL clinical trial (see Bargou et al., cited above), a total of 39patients have been treated until August 2008. At this time point, it hasbeen found that all patients with a neurological reaction that led topermanent discontinuation of the CD19×CD3 bispecific antibody treatmenthad a low B:T cell ratio (i.e., a B:T cell ratio threshold below 1:5).In particular, 5 neurological reactions have been observed in 10patients with low B:T cell ratio (5/10), while no patient with a highB:T cell ratio (i.e., a B:T cell ratio higher than 1:5) had aneurological reaction that would have led to permanent discontinuationof CD19×CD3 bispecific antibody treatment (0/29).

Thereafter, a specific cohort for patients with low B:T cell ratio, i.e.an increased risk for early neurological reactions, was established inorder to prospectively analyze the outlined theory and to specificallyfind mitigation steps for the patients at increased risk.

Since establishing these separated cohorts for high risk patients, 8 NHLpatients were prospectively treated (data as of July 2009): 6 patientswith low B:T cell ratio, 2 patients with high B:T cell ratio. Again nopatient with a high B:T cell ratio had a neurological reaction, while 3out of 6 patients with a low B:T cell ratio had a neurological reaction,leading to discontinuation of the treatment.

In sum, 69 NHL patients, including B-cell chronic lymphocytic leukemia(CLL) and mantle cell lymphoma (MCL), have been treated with a CD19×CD3bispecific antibody, both with bolus infusion and continuous infusion:

Neurological reactions have been observed in 61% of the patients withlow B:T cell ratio. In contrast, only 2% of the patients with high B:Tcell ratio showed such adverse events (see the following examples).

In another clinical trial phase II study, 15 μg of CD19×CD3 bispecificsingle chain antibody per square meter patient body surface area per dayhave been administered to adult ALL patients by continuous infusion forat least four weeks. One out of 11 ALL patients of the high risk grouphaving a B:T cell ratio below 1:5 showed a neurological reaction,leading to discontinuation of the treatment. In contrast, none of the 6patients of the low risk group having a B:T cell ratio higher than 1:5showed a neurological reaction.

Moreover, in a retrospective analysis of 39 NHL patients, a baseline Bcell to T cell (B:T) ratio in peripheral blood at or below 1:5 to 1:10was identified as the only predictive factor for the subsequentoccurrence of neurological AEs. The predictive value was thenprospectively confirmed in 8 additional patients (see Example 1).

In sum, these data establish a low B:T cell ratio, i.e., a B:T cellratio of about 1:5 or lower as a potential high risk factor for theoccurrence of adverse effects including neurological reactions in thetreatment of malignant CD19 positive lymphocytes occurring in leukemiaand lymphoma such as NHL, MCL, CLL and ALL in patients who are treatedwith a CD19×CD3 bispecific antibody (see Examples 1 and 4).

Thus, it was an aim of the present invention to provide a method thatallows identifying patients who may be at a risk of suffering fromadverse effects when being treated with a CD19×CD3 bispecific antibody.This method will improve drug compliance, since the identification ofpatients who are at a risk of suffering from adverse effects allowsadjusting the dosage regimen of the CD19×CD3 bispecific antibody. Infact, the present inventors have applied their finding that a B:T cellratio of about 1:5 or lower could be a potential risk factor forsuffering from adverse effects in the treatment with a CD19×CD3bispecific antibody and have thus developed a dosage regimen which isintended to prevent and/or ameliorate these adverse effects.

Accordingly, in a first aspect the present invention provides a methodfor assessing (analyzing) the risk of potential adverse effects for ahuman patient mediated by the administration of a CD19×CD3 bispecificantibody to said patient comprising determining in a sample from saidpatient the ratio of B cells to T cells of said patient, wherein a ratioof about 1:5 or lower is indicative for a risk of potential adverseeffects for said patient.

“Assessing (analyzing) the risk” means that the method of the firstaspect of the present invention aims at assessing or analyzing as towhether or not a patient has a higher or lower likelihood or probability(i.e., an increased or decreased risk, respectively) to encounteradverse effects. Accordingly, as is commonly known, a risk does notnecessarily mean that a patient will or will not encounter adverseeffects.

In the present invention, when a patient has a B:T cell ratio of about1:5 or lower, said patient has (is at) an increased risk of potentialadverse effects, also including the onset of an adverse effect, while apatient who has a B:T cell ratio higher than 1:5 does not have (is notat) or at least has (is at) a decreased risk of potential adverseeffects, also including the onset of an adverse effect.

Accordingly, a B:T cell ratio of about 1:5 or lower is indicative for arisk of adverse effects, while a B:T cell ratio of higher than 1:5 isnot indicative for a risk of adverse effects.

Thus, the term “indicative for” when used in the context of the methodof the first aspect of the present invention means that a patient has anincreased risk of potential adverse effects if the B:T cell ratio isabout 1:5 or lower or has a decreased risk of potential adverse effectsif the B:T cell ratio is higher than 1:5.

An “adverse effect” is a harmful and undesired effect resulting frommedication in the treatment of a patient with a CD19×CD3 bispecificantibody. An adverse effect may also be termed a “side effect”. Someadverse effects only occur only when starting, increasing ordiscontinuing a treatment. The inventors have observed that the adverseeffect seen in the treatment of patients with a CD19×CD3 bispecificantibody occurred after about 12 to 120 hours after the start of thetreatment and are reversible.

An adverse effect may cause medical complications. The inventors haveobserved neurological reactions in patients treated with a CD19×CD3bispecific antibody. These neurological reaction, unless they can bestopped or avoided, lead to non-compliance with the CD19×CD3 bispecificantibody treatment.

However, as mentioned herein, the inventors found that the B:T cellratio is an indicator as to whether or not patients are at a risk ofpotential adverse side effects. Specifically, a B:T cell ratio about orlower 1:5 is an indicator that patients are at a risk of potential sideeffects, while a B:T cell ratio higher than about 1:5 is an indicatorthat patients have no or at least have a decreased risk of potentialside.

As mentioned before, the method of the first aspect of the presentinvention is for assessing (analyzing) the risk of adverse effects and arisk includes the assessment/analysis of likelihood or a probability.Accordingly, the term “potential” when used in the context of adverseeffects means that—though a patient may have a B:T cell ratio of about1:5 or lower—said patient does not necessarily have to encounter adverseeffects.

Likewise, though a patient may have a B:T cell ratio higher than about1:5—said patient does not necessarily have to not encounter adverseeffects. Accordingly, the term “potential” implies that the method ofthe first aspect of the present invention provides predictions as towhether or not a patient may encounter adverse effects,but—self-explanatory as it is—cannot provide a 100% safe prediction,since, apart from the B:T cell ratio individual factors such as sex,age, weight, nutritional status, health status, pre-medication etc. mayhave an influence as to whether or not a patient will encounter adverseeffects.

In accordance with the present invention an adverse effect is preferablycharacterized by a neurological reaction (also sometimes referred toherein as “CNS reaction” or “CNS event”, for which reason these termscan be equally used). Said neurological reaction is preferably one ormore selected from the group consisting of: confusion, ataxia,disorientation, dysphasia, aphasia, speech impairment, cerebellarsymptoms, tremor, apraxia, seizure, grand mal convulsion, palsy, andbalance disorder.

The degree of an adverse effect can, for example, be measured inaccordance with the NCI Common Terminology Criteria for Adverse Eventsv3.0 (CTCAE) (Publish Date: Dec. 12, 2003) in grades. A Grade refers tothe severity of the adverse effects. The CTCAE v3.0 displays grades 1through 5 with unique clinical descriptions of severity for each adverseeffects:

Grade 1: mild adverse effects

Grade 2: Moderate adverse effects

Grade 3: Severe adverse effects

Grade 4: Life-threatening or disabling adverse effects.

Grade 5: Death of the patient.

A “patient” is a human individual who will be or is treated with aCD19×CD3 bispecific antibody. In accordance with the present invention,the patient is suspected/assumed to comprise or already comprisesmalignant CD19 positive lymphocytes (in particular B cells). In thelatter case, said patient has already been diagnosed to comprise suchcells. These malignant CD19 positive lymphocytes (in particular B cells)are present in a patient developing and/or suffering from leukemiaand/or lymphoma. In accordance with the present invention a patient isthus in need of a treatment of malignant CD19 positive lymphocytes.Preferably, a patient who will be or is treated with a CD19×CD3bispecific antibody is (or has been) diagnosed in accordance with themethod of the first aspect of the invention as described herein.

“Mediated by” when used in the context of the method of the first aspectof the present invention means that adverse effects that a patient mayor may not encounter are caused by the administration of a CD19×CD3bispecific antibody. Put it differently, the CD19×CD3 antibody is thecausative agent that may cause potential adverse effects in a patient.

The administration may be in the form of a bolus administration orcontinuous administration, with continuous administration beingpreferred.

In accordance with the present invention by the term “sample” isintended any biological sample obtained from a human patient containingpolynucleotides or polypeptides or portions thereof. Biological samplesinclude body fluids (such as blood, serum, plasma, urine, saliva,synovial fluid and spinal fluid) and tissue sources found to malignantCD19 positive lymphocytes. Methods for obtaining tissue biopsies andbody fluids from patients are well known in the art. Generally, abiological sample which includes peripheral blood mononuclear cells(PBMCs), in particular B cells and T cells is preferred as a source.

A sample which includes peripheral blood mononuclear cells (PBMCs), inparticular B cells and T cells is preferably taken from peripheral bloodof a human patient.

Other preferred samples are whole blood, serum, plasma or synovialfluid, with plasma or serum being most preferred. However, a sample fromperipheral blood of a human patient is particularly preferred.

A “B:T cell ratio” as used herein refers to the ratio of the number of Bcells and the number of T cells. It is preferably determined in a sampletaken from a human patient. Preferably, the sample is taken from theperipheral blood of a human patient. The number of B or T cells, forexample, in a peripheral blood sample can be determined by any meansusually applied in the art, for example, by FACS analysis.

The B:T cell ratio is preferably about 1:5 or lower including a B:T cellratio of about 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12; 1:13, 1:14, 1:15,1:20, 1:100, 1:200, 1:400, 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000or even lower, with 1:9 1:10, 1:50, 1:100, 1:500, 1:1000 beingpreferred, with 1:9 being particularly preferred.

“Determining the B:T cell ratio” includes

-   (a) determining the total B cell number in a sample from a patient,    preferably in a peripheral blood sample of the patient;-   (b) determining the total T cell number in sample from a patient,    preferably in a peripheral blood sample of the patient;-   (c) calculating the ratio of the B cell number of step (a) and the T    cell number of step (b) in order to obtain a B:T cell ratio.

Of note, a low B:T cell ratio can also be seen as high T:B ratio; andvice versa. Accordingly, the ratios provided herein for a low B T cellratio would then have to be reversed.

In contrast, patients showing a B:T cell ratio higher than about 1:5(preferably 1:9), including a B:T cell ratio of higher than about 1:4,1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or higher,have a decreased risk of suffering from potential adverse effects uponadministration of a CD19×CD3 bispecific antibody.

Accordingly, the present invention also envisages a method for assessing(analyzing) the risk of potential adverse effects for a human patientmediated by the administration of a CD19×CD3 bispecific antibody to saidpatient comprising determining in a sample from said patient the ratioof B cells to T cells of said patient, wherein a ratio of higher thanabout 1:5 (preferably 1:9) is indicative for a decreased risk ofpotential adverse effects for said patient.

Having observed that patients who have a B:T cell ratio of about 1:5 orlower are at an increased risk of potential adverse effects, theinventors developed a concept that allows the treatment of thesepatients with a CD19×CD3 bispecific antibody. Bearing this in mind, ithas been elucidated that the T cells of such high risk patients have tobe pre-adapted or partially activated by the administration of a lowdose of antibody for several days before the dose can then be escalated.So it has been found that a significant decrease in dose given per timeunit potentially increases tolerability to said antibody in the highrisk patients.

In essence, the inventors found that “adapting” a patient to a CD19×CD3bispecific antibody prior to the therapy with a CD19×CD3 bispecificantibody is beneficial for avoiding undesired adverse effect(particularly the unwanted neurological reactions) (see Examples 6 and7).

Accordingly, the present invention relates in a second aspect to amethod (dosage regimen) for administering a CD19×CD3 bispecific antibodyto a human patient having a B:T cell ratio of about 1:5 or lower,comprising:

-   (a) administering a first dose of said antibody for a first period    of time; and consecutively-   (b) administering a second dose of said antibody for a second period    of time;    wherein said second dose exceeds said first dose.

It will be understood that in the context of the present invention, theterm “method” includes a “dosage regimen” to be used in a method of thepresent invention.

In the context of the present invention “administration of a CD19×CD3bispecific antibody” or “administering a CD19×CD3 bispecific antibody”or any other grammatical form thereof means that the CD19×CD3 antibodyis in the form of a pharmaceutical composition, optionally comprising apharmaceutically acceptable carrier. Accordingly, it is to be understoodthat a pharmaceutical composition comprising a CD19×CD3 bispecificantibody is administered to a human patient.

The term “administering” in all of its grammatical forms meansadministration of a CD19×CD3 bispecific antibody (in the form of apharmaceutical composition) either as the sole therapeutic agent or incombination with another therapeutic agent.

It is thus envisaged that the pharmaceutical composition of the presentinvention are employed in co-therapy approaches, i.e. inco-administration with other medicaments or drugs, for example, othermedicaments for treating malignant CD19 positive lymphocytes in apatient and/or any other therapeutic agent which might be beneficial inthe context of the methods of the present invention.

For example, if the methods of the invention are carried out for thetreatment of B-lineage acute lymphoblastic leukemia or aggressive NHL,it can advantageously be combined with inthrathecal chemotherapy inorder to eliminate target B cells from the CNS. For example, theinthratecal chemotherapy could be performed prior to the administrationof the CD19×CD3 bispecific single chain antibody according to themethods described herein.

The administration of a pharmaceutical composition referred to herein ispreferably an intravenous administration. It follows that in the methodsof the present invention the route of administration in step (a) and/orthe route of administration in step (b) is intravenous. It may beadministered as a bolus injection or continually (continuously), withcontinually being preferred.

The administration of a CD19×CD3 bispecific antibody (for example in theform of a pharmaceutical composition) can be a bolus injection orcontinually or as also sometimes used herein continuously, withcontinually or continuously being preferred. A continual administrationrefers to an administration which is essentially without interruption.“Essentially without interruption” includes a continual administrationusually without an uninterrupted flow or spatial extension.

In some embodiments, said first dose is not therapeutically active, i.e.it is a subtherapeutic dose. Without being strictly bound, for thepurpose of the present invention a dose of 5 μg/m²/d or lower is held tobe subtherapeutic.

In a preferred embodiment of the present invention the second dose istherapeutically active.

By “therapeutically effective amount” or “therapeutically active” ismeant a dose of a CD19×CD3 bispecific antibody that produces thetherapeutic effects for which it is administered.

The exact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art and described above, adjustments for age, body weight,general health, sex, diet, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. The therapeutic effect ofthe respective methods or method steps of the present invention isadditionally detectable by all established methods and approaches whichwill indicate a therapeutic effect. It is, for example, envisaged thatthe therapeutic effect is detected by way of surgical resection orbiopsy of an affected tissue/organ which is subsequently analyzed by wayof immunohistochemical (IHC) or comparable immunological techniques.Alternatively it is also envisaged that the tumor markers in the serumof the patient (if present) are detected in order to diagnose whetherthe therapeutic approach is already effective or not. Additionally oralternatively it is also possible to evaluate the general appearance ofthe respective patient (fitness, well-being, decrease of tumor-mediatedailment etc.) which will also aid the skilled practitioner to evaluatewhether a therapeutic effect is already there. The skilled person isaware of numerous other ways which will enable him or her to observe atherapeutic effect of the compounds of the present invention.

In a third aspect, the present invention relates to a method fortreating malignant CD19 positive lymphocytes in a human patient having aB:T cell ratio of about 1:5 or lower, said method comprising:

-   (a) administering a first dose of a CD19×CD3 bispecific antibody for    a first period of time; and consecutively-   (b) administering a second dose of said antibody for a second period    of time;    wherein said second dose exceeds said first dose.

Malignant CD19 positive lymphocytes (in particular B cells) are found inleukemia and/or lymphoma. Accordingly, the CD19 positive lymphocytes arein a preferred embodiment lymphoma or leukemia cells.

“Malignant” describes lymphocytes (in particular B cells) thatcontribute to a progressively worsening disease, in particular lymphomaor leukemia and the diseases described herein. The term is most familiaras a description of cancer, here lymphoma and leukemia and the diseasesdescribed herein. Malignant CD19 positive lymphocytes (in particular Bcells) are not self-limited in their growth, are capable of invadinginto adjacent tissues, and may be capable of spreading to distanttissues (metastasizing). Malignant when used herein is synonymous withcancerous.

However, as “normal” (non-malignant) lymphocytes (in particular B cells)also express CD19, it is to be expected that the CD19×CD3 bispecificantibody also binds these normal lymphocytes (in particular B cells) andupon recruiting cytotoxic T cells (because of the second specificity ofthe bispecific CD19×CD13 antibody) depletes these normal B cells. Yet,it is expected that the population of these normal B cells isreconstituted in the absence of the CD19×CD3 bispecific antibody. It wasobserved by Leandro and co-workers that after their depletion by ananti-CD20 antibody, B cells were reconstituted in rheumatoid arthritispatients (Arthritis Rheum. 2006 February; 54(2):613-20). As CD20,likewise CD19 is expressed on almost all B cells, it can be expectedthat B cells upon depletion by the bispecific CD19×CD3 antibody arereconstituted, too.

The lymphoma is preferably indolent or aggressive B cell non-Hodgkinlymphoma (B NHL), mantle cell lymphoma (MCL) or chronic lymphaticleukemia (CLL). Within the meaning of the invention, the term “B cellnon-Hodgkin lymphoma” or “B cell derived non-Hodgkin lymphoma” comprisesboth indolent and aggressive B cell non-Hodgkin lymphoma (B NHL). Theterm “indolent or aggressive B cell non-Hodgkin lymphoma (B NHL)” asused herein represents malignant B cell-derived tumorous diseases.Indolent B NHL are low malignant lymphomas. Aggressive B-NHL are highmalignant lymphomas. The B cell non-Hodgkin lymphoma (B NHL) mayadvantageously be a follicular lymphoma, lymphoplasmacytic lymphoma,marginal zone cell lymphoma, mantle cell lymphoma (MCL), diffuse large Bcell lymphoma (DLBCL), Burkitt's lymphoma, small lymphocytic lymphoma(SLL/CLL) and any other B cell derived subtype. The term “B cellleukemia” as used herein may advantageously be any B cell leukaemia(e.g. chronic lymphocytic leukaemia or acute lymphocytic leukaemia). Forfurther reference see e.g. http colon-backslash-backslashwww.cancer.org. Preferably, indolent non-Hodgkin B cell lymphoma may betreated with a bispecific single chain antibody directed against bothhuman CD3 and human CD19 as demonstrated in the following examples.

The leukemia is preferably B-lineage acute lymphoblastic leukemia (ALL).

In a fourth aspect, the present invention relates to a method forameliorating and/or preventing an adverse effect mediated by theadministration of a CD19×CD3 bispecific antibody to a human patienthaving a B:T cell ratio of about 1:5 or lower, said method comprising:

-   (a) administering a first dose of said antibody for a first period    of time, and consecutively-   (b) administering a second dose of said antibody for a second period    of time;    wherein said second dose exceeds said first dose.

The adverse effect is preferably a neurological reaction, preferably oneor more selected from the group consisting of: confusion, ataxia,disorientation, dysphasia, aphasia, speech impairment, cerebellarsymptoms, tremor, apraxia, seizure, grand mal convulsion, palsy, andbalance disorder (see also Examples 2 and 3).

Specifically, neurological reactions observed during the starting phaseof treatment with the CD19×CD3 bispecific antibody include for exampleconfusion and disorientation. “Confusion” as used herein refers to lossof orientation which is the ability to place oneself correctly in theworld by time, location, and personal identity, and often memory whichis the ability to correctly recall previous events or learn newmaterial. The patients usually have difficulties to concentrate andthinking is not only blurred and unclear but often significantly sloweddown. Patients with neurological reactions also suffer from loss ofmemory. Frequently, the confusion leads to the loss of ability torecognize people and/or places, or to tell time and the date. Feelingsof disorientation are common in confusion, and the decision-makingability is impaired. Neurological reactions further comprise blurredspeech and/or word finding difficulties. This disorder may impair both,the expression and understanding of language as well as reading andwriting. Besides urinary incontinence, also vertigo and dizziness mayaccompany neurological reactions in some patients.

The occurrence of neurological reactions in the treatment of B celldependent lymphatic or leukemic malignancies with the CD19×CD3bispecific antibody may be further influenced by the following factors:

1. Presence of Drug

The CD19×CD3 bispecific antibody retargets T cell cytotoxicity tomalignant CD19 positive lymphocytes present, for example, in B celllymphoma or leukemia cells. In light of this, it can be reasonablyassumed that it is the presence of CD19×CD3 bispecific antibody in thebody of a patient which is responsible for the adverse effects.Furthermore, side effects are observed only in parts of the body wherethe CD19×CD3 bispecific antibody is biologically active. Accordingly,neurological reactions upon treatment with the CD19×CD3 bispecificantibody are assumed to be dependent on the presence of said antibody inthe cerebrospinal fluid (CSF; liquor) of the patient. This may besupported by the fact that the CD19×CD3 bispecific antibody as well as Tcells have only been found in the CSF of NHL patients with a low B:Tcell ratio. As explained herein, this patient population has anincreased risk for the development of neurological reactions uponantibody treatment. This finding may suggest that the CD19×CD3bispecific antibody is able to enter the perivascular space dividing theblood vessels and the CNS (including the brain), in high risk NHL andALL patients. There, the CD19×CD3 bispecific antibody may then engage Tcells to target local B cells (either benign or malign) which possiblyleads to local cytokine release which in turn could cause neurologicalreactions.

2. Drug Dose

Further, the neurological reactions seem to be dependent on the dose ofthe CD19×CD3 bispecific antibody. For example, neurological reactionshave not been observed upon continuous administration of 5 μg/m² bodysurface area of CD19×CD3 bispecific antibody, but with 15 μg/m² bodysurface area or more CD19×CD3 bispecific antibody in the high risk groupof patients. For this reason, as mentioned herein, a dose of less than 5μg/d/m² is deemed to be subtherapeutic. The effect of the drug dose isevident from the data shown in the appended examples. This observationmay imply a dose dependency of neurological reactions in high riskpatients with low B:T cell ratio.

3. Presence of Target Cells and Effector Cells

As set forth above, the neurological reactions upon CD19×CD3 bispecificantibody-treatment are assumed to depend on the presence of i) targetcells, i.e. CD19-antigen carrying B cells and ii) effector cells, i.e.cytotoxically active T cells carrying the CD3 antigen, in the PVS/CNS.

In view of this, it is intriguing to hypothesize that the depletion ofe.g. the target B cell from the PVS/CNS should result in the avoidanceof neurological reactions. In fact, this is exactly what has beenobserved in the mentioned phase II study in which B lineage acutelymphoblastic leukemia (ALL) patients are being currently treated withthe CD19×CD3 bispecific antibody:

In ALL, there is generally a high incidence of leukemic lesions in theCNS. Therefore, each of the ALL patients enrolled in the clinical phaseII study referred to herein had received standard ALL therapies in thepast, including methotrexate i.v. and/or intrathecal chemotherapy, inorder to prevent central nervous system relapses. Some of them receivedin addition irradiation of the neuroaxis. The ALL patients thereafterreceived a consolidation therapy, i.e. they obtained several fourweek-treatment cycles of continuous administration of 15 μg/d/m² ofCD19×CD3 bispecific antibody. Only one of the thus far enrolled 17 ALLpatients who have been treated with the CD19×CD3 bispecific antibody hasdeveloped neurological reactions. This patient was one out of 11patients belonging to the high risk group having a B:T cell ratio lowerthan 1:5. None of the six patients of the low risk group with a B:T cellratio higher than 1:5 showed neurological reactions. It is thereforehypothesized that the mentioned (pre-symptomatic) central nervous system(CNS) treatment reduced the risk of a neurological reaction in the ALLpatient in that the B lymphocytic target cells have been removed fromthe PVS and CNS, including the brain. However, in the absence of Btarget cells in these tissues, there is no full activation of thecytotoxic T cells. Therefore, less frequent neurological reactions couldbe observed in said patient populations.

Accordingly, the absence of one of the above factors, in the mentionedcase the presence of target B cells in the PVS/CNS, could possibly helpto prevent neurological reactions. However, for example, intrathecalchemotherapy is not the therapy of choice in NHL treatment. Forinstance, it is not effective in indolent NHL therapy, and it is not yetknown whether it could be a treatment option for aggressive NHL. Inaddition, intrathecal chemotherapy is highly toxic for ALL patients andtherefore associated with considerable health risks.

In light of the above, the depletion of any one of the above indicatedfactors without loosing therapeutic efficacy is no trivial task since itis for example not easily possible to avoid the presence of B cells inthe PVS/CNS of NHL. Furthermore, it has also been found that othermeasures, including the pre- or co-administration of high doses ofsteroids could not prevent neurological reactions in the high riskpatients.

However, by way of applying the methods/dosage regimens of the presentinvention, it is possible to ameliorate and/or prevent adverse effectsfor patients who are at an increased risk of such adverse effects ifthey have a B:T cell ratio of about 1:5 or lower. The present inventionenvisages providing dosage regimens (methods) which are even independentof the above mentioned factors that could influence a treatment with aCD19×CD3 bispecific antibody.

Thus, the present invention in a preferred aspect relates to a methodfor assessing (analyzing) the risk of potential adverse effects for ahuman patient mediated by the administration of a CD19×CD3 bispecificantibody to said patient comprising determining the ratio of B cells toT cells determining in a sample from said patient, wherein a ratio ofabout 1:5 or lower is indicative for a risk of potential adverse effectsfor said patient, wherein said patient is

-   (a) administered a first dose of said antibody for a first period of    time; and is consecutively-   (b) administered a second dose of said antibody for a second period    of time;    -   wherein said second dose exceeds said first dose;        for-   (i) treating malignant CD19 positive lymphocytes; and/or-   (ii) for ameliorating and/or preventing an adverse effect mediated    by the administration of a CD19×CD3 bispecific antibody.

Preferably, in this preferred aspect, the patient is administered athird dose of said antibody for a third period of time as described inherein below. Accordingly, the embodiments and aspects described hereinin the context of the three-stage method (dose regimen) are applicableto this preferred aspect.

In one aspect of the methods of the present invention said second periodof time exceeds said first period of time. The term “exceeds” means thatthe second period of time is at least one day longer than the firstperiod of time.

Each of the methods (dosage regimens) of the present invention can berepeated, for example, for one, two, three, four, five, six, or moretimes and in any event as often as there is a beneficial effect for apatient in ameliorating and/or treating malignant CD19 positivelymphocytes, thereby treating lymphoma or leukemia. Dependent on theratio of the B:T cell ratio of a patient, in accordance with theteaching of the present invention the practitioner can decide as towhether the patient has to be “adapted” to a further treatment with aCD19×CD3 bispecific antibody prior by applying the dosage regimens ofthe present invention (i.e., administering a low dose of a CD19×CD3bispecific antibody prior to administering a higher dose in order to“adapt” the patient).

It must be understood that the dose or day ranges given herein areillustrated by increments of one, two, three, four or five. Theseranges, however, in case of increments higher than one also encompasssmaller increments, for example those exemplified by increments of one(10 to 30 includes for example 10, 11, 12, 13, 13 etc. up to 30), orstill smaller increments, for example values after the decimal point.

In another aspect of the present invention, it is envisaged that saidfirst period of time is at least 3 days long, whereby even longerperiods of time of for example 8, 9, 10, 11, 12, 13 or 14 days are notexcluded. “Longer” is thereby not limited to a (one) complete day as thelowest time unit, i.e. ½ days, or fully hours are also conceivable. Itis however preferred that the smallest time unit is one full day.

Accordingly, said first period of time exceeds 3 days. More preferably,it is envisaged that said first period of time is between 3 days and 10days, with 7 days being particularly preferred.

As used herein, a time interval which is defined as “X to Y” equateswith a time interval which is defined as “between X and Y”. Both timeintervals specifically include the upper limit and also the lower limit.This means that for example a time interval “3 to 10 days” or between “3to 10 days” includes a period of time of one, two, three, four, five,six, seven and/or eight days.

As mentioned herein, the inventors observed that “adapting” a humanpatient having a B:T cell ratio of about 1:5 or lower to the treatmentwith a CD19×CD3 bispecific antibody during a first period of time allowsthe treatment of the human patient with an increased second dose of theantibody for a second period of time, whereby adverse effects (inparticular neurological reactions) can be better controlled, i.e., couldbe avoided or at least kept within an acceptable grade in accordancewith the CTCAE.

However, for achieving this improvement it is required to “adapt” thehuman patient having a B:T cell ratio of about 1:5 or lower to theCD19×CD3 bispecific antibody by administering a first dose of theantibody for a first period of time (wherein said first dose is lowerthan the consecutive (second) dose). The administration can be a bolusinjection or a continuous administration, whereby a continuousadministration is preferred.

Likewise the duration of the first period of time, the duration of thesecond period of time may be variable in view of, for example, the age,sex, body weight, etc. of the human patient.

Accordingly, in another aspect of the present invention, it is envisagedthat said second period of time is at least 18 days long, whereby evenlonger periods of time of for example 19, 20, 25, 30, 35, 40, 45, 49,50, 55, 60, 65, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 90days are not excluded. “Longer” is thereby not limited to a (one)complete day as the lowest time unit, i.e. ½ days, or fully hours arealso conceivable. It is however preferred that the smallest time unit isone full day.

Accordingly, said second period of time exceeds 18 days. Morepreferably, it is envisaged that said second period of time is between18 days and 81 days, with 21 or 49 days being particularly preferred.

As used herein, a time interval which is defined as “X to Y” equateswith a time interval which is defined as “between X and Y”. Both timeintervals specifically include the upper limit and also the lower limit.This means that for example a time interval “18 to 81 days” or between“18 to 81 days” includes a period of time of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,61, 62, 63 and/or 64 days.

In a more preferred embodiment of the methods/dosage regimens of thepresent invention, said first period of time is between 3 days and 10days, and said second period of time is between 18 and 81 days.

In an even more preferred embodiment, said first period of time is 7days and said second period of time is 21 or 49 days.

In the clinical trials mentioned herein, it was observed that a dose of15 μg/m²/d in the treatment of NHL effected tumor shrinkage as could bevisualized in computer tomography. It was also observed that a dose of15 μg/m²/d in the treatment of ALL resulted in minimal residual diseaseand could even eliminate MRD.

Minimal residual disease (MRD) is the name given, to small numbers ofleukemic/lymphoma cells that remain in the patient during treatment orafter treatment when the patient is in remission (no symptoms or signsof disease). Up until a decade ago none of the tests used toassess/detect cancer, were sensitive enough to detect MRD. Now, however,very sensitive molecular biology tests are available—based on DNA, RNAor Proteins—and these can measure minute levels of cancer cells intissue samples, sometimes as low as 1 cancer cell in million normalcells.

In cancer treatment, particularly leukaemia, MRD testing has severalimportant roles: determining whether treatment has eradicated the canceror whether traces remain, comparing the efficacy of differenttreatments, monitoring patient remission status and recurrence of theleukemia or cancer and choosing the treatment that will best meet thoseneeds (personalization of treatment)

Accordingly, in a further aspect of the methods/dosage regimens of thepresent invention, said first dose is between 1 and 15 μg/m²/d, i.e. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μg/m²/d. Particularlypreferred is a dose of 5 or 15 μg/m²/d.

As used herein, a dose interval which is defined as “between X and Y”equates with a dose interval which is defined as “X to Y”. Both doseintervals specifically include the upper limit and also the lower limit.This means that for example a dose interval “between 1 and 15” or “1 to15” includes a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15μg/m²/d.

“d” denotes one day.

“m²” denotes a square meter of a patient's body surface (BSA). The“normal” average BSA is generally taken to be about 1.73 m² for anadult, for a neonate it is about 0.25 m², for a 2 year old child it isabout 0.5 m², for a 9 year old child it is about 1.07 m², for a 10 yearold child it is about 1.14 m², for a 12-13 year old child it is about1.33 m², for men it is about 1.9 m² and for women it is about 1.6 m².

However, the BSA can also be calculated more precisely by one of thefollowing formulas (each of these formulas can be applied whencalculating the BSA):

-   -   The Mosteller formula (Mosteller, N Engl J Med 1987 Oct. 22;        317(17): 1098):    -   BSA (m²)=([Height (cm)× Weight (kg)]/3600)^(1/2) or in inches        and pounds:    -   BSA (m²)=([Height (in)× Weight (lbs)]/3131)^(1/2)    -   The DuBois formula (DuBois, Arch Int Med 1916 17:863-871):    -   BSA (m²)=0.007184× Height (cm)^(0.725)× Weight (kg)^(0.425)    -   The Haycock formula (Haycock, The Journal of Pediatrics 1978        93:1: 62-66):    -   BSA (m²)=0.024265× Height (cm)^(0.3964)× Weight (kg)^(0.5378)    -   The Gehan formula (Gehan, Cancer Chemother Rep 1970 54:225-35):    -   BSA (m²)=0.0235× Height (cm)^(0.42246)× Weight (kg)^(0.51456)    -   The Boyd formula (Boyd, University of Minnesota Press, 1935)    -   BSA (m²)=0.0003207× Height (cm)^(0.3)× Weight        (grams)^((0.7285−(0.0188×log 10(grams))

The term “μg” includes “μg of the CD19×CD3 bispecific antibodypreparation”. It is preferred that not more than 10% of said CD19×CD3bispecific antibody preparation is incorrectly folded. It follows thatin a preferred embodiment, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or even 100% of the CD19×CD3 bispecific antibody is correctlyfolded. It is also conceivable that the antibody preparation mayoptionally comprise further ingredients, for example a lyoprotectant, asurfactant, a filler, a binder, and/or bulking agent etc. The amount ofsuch further ingredients is, preferably, not included in the term “μg”as used in the context of the “dose” and/or methods (dosage regimens) ofthe present invention.

A dose of, for example, 1 μg/m²/d means that 1 μg of the CD19×CD3bispecific antibody is administered evenly or continuously across oneday per square meter body surface. “Continuously across one day” refersto an infusion which is allowed to proceed permanently withoutinterruption.

In a further aspect of the methods/dosage regimen of the presentinvention, said second dose is between 15 and 60 or 15 and 90 μg/m²/d,i.e. 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 μg/m²/d or 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 70, 80 and 90 μg/m²/d. Particularlypreferred is a dose of 60 or 90 μg/m²/d. Said second dose is thustherapeutically active.

In a preferred embodiment, said first dose is between 5 and 15 μg/m²/dand said second dose is between 15 and 60 or 15 and 90 μg/m²/d.

As used herein, a dose interval which is defined as “between X and Y”equates with a dose interval which is defined as “X to Y”. Both doseintervals specifically include the upper limit and also the lower limit.This means that for example a dose interval “between 15 and 60” or “15to 60” includes a dose of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and/or 60μg/m²/d.

It must be understood that the ranges given herein are illustrated byincrements of five. These ranges, however, also encompass smallerincrements, for example those exemplified by increments of one (10 to 30includes for example 10, 11, 12, 13, 13 etc. up to 30), or still smallerincrements, for example values after the decimal point.

Preferably, not included in the methods for administering a CD19×CD3bispecific antibody, for treating malignant CD19 positive lymphocytes,or for ameliorating and/or preventing an adverse effect mediated by theadministration of a CD19×CD3 bispecific antibody are the followingadministration schemes:

-   (i) 5 μg/m² of the bispecific antibody for one day followed by    administration of 15 μg/m² as daily dose for the remaining period    (second and each further consecutive day); and/or-   (ii) 15 μg/m² of the bispecific antibody for one day followed by    administration of 45 μg/m² as daily dose for the remaining period    (second and each further consecutive day); and/or-   (iii) 5 μg/m² of the bispecific antibody for one day followed by    administration of 15 μg/m² for one day, followed by administration    of 45 μg/m² as daily dose for the remaining period (third and each    further consecutive day); and/or-   (iv) less than 10-80 μg/m² of the bispecific antibody for one day    followed by administration of a dose of 10-80 μg/m² (second and each    further consecutive day); and/or-   (v) less than 10-80 μg/m² of the bispecific antibody for one day    followed by administration of a dose of less 10-80 μg/m² for one    day, followed by administration of a dose of less 10-80 μg/m² (third    and each further consecutive day).

As mentioned herein, patients having a B:T cell ratio higher than 1:5,do not necessarily have to be adapted to the treatment with a CD19×CD3bispecific antibody by way of the dosage regimen of the presentinvention. These patients having a decreased risk of potential adverseeffects could be treated by administration of a CD19×CD3 bispecificantibody in a constant dose of 5 μg to 75 μg per square meter bodysurface area per day for at least four weeks. The administration ispreferably a continuous administration.

In another embodiment of the methods (dosage regimen) of the presentapplication, said methods further comprise administering after a firstand second dose for a first and second period of time a third dose ofsaid antibody for a third period of time. Accordingly, the presentinvention provides a three-stage method (dosage regimen).

The administration of said third dose is intravenously. It can beadministered in the form of a bolus injection or continuously, withcontinuously being preferred.

In one aspect of the methods of the present invention said third periodof time exceeds said first and second period of time. The term “exceeds”means that the third period of time is at least one day longer than thefirst and second period of time.

Likewise the duration of the first and second period of time, theduration of the third period of time may be variable in view of, forexample, the age, sex, body weight, etc. of the human patient.

In the three-stage dosage regimen aspect of the present invention, it isenvisaged that said first period of time is at least 3 days long,whereby even longer periods of time of for example 8, 9, 10, 11, 12, 13or 14 days are not excluded. “Longer” is thereby not limited to a (one)complete day as the lowest time unit, i.e. ½ days, or fully hours arealso conceivable. It is however preferred that the smallest time unit isone full day.

Accordingly, said first period of time exceeds 3 days. More preferably,it is envisaged that said first period of time is between 3 days and 10days, with 7 days being particularly preferred.

As used herein, a time interval which is defined as “X to Y” equateswith a time interval which is defined as “between X and Y”. Both timeintervals specifically include the upper limit and also the lower limit.This means that for example a time interval “3 to 10 days” or between “3to 10 days” includes a period of time of one, two, three, four, five,six, seven and/or eight days.

In the three-stage dosage regimen aspect of the present invention, it isenvisaged that said second period of time is at least 3 days long,whereby even longer periods of time of for example 8, 9, 10, 11, 12, 13or 14 days are not excluded. “Longer” is thereby not limited to a (one)complete day as the lowest time unit, i.e. ½ days, or fully hours arealso conceivable. It is however preferred that the smallest time unit isone full day.

Accordingly, said first period of time exceeds 3 days. More preferably,it is envisaged that said first period of time is between 3 days and 10days, with 7 days being particularly preferred.

As used herein, a time interval which is defined as “X to Y” equateswith a time interval which is defined as “between X and Y”. Both timeintervals specifically include the upper limit and also the lower limit.This means that for example a time interval “3 to 10 days” or between “3to 10 days” includes a period of time of one, two, three, four, five,six, seven and/or eight days.

In the three-stage dosage regimen aspect of the present invention, it isenvisaged that said third period of time is at least 8 days long,whereby even longer periods of time of for example 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70 and/or 71 days are not excluded. “Longer” isthereby not limited to a (one) complete day as the lowest time unit,i.e. ½ days, or fully hours are also conceivable. It is howeverpreferred that the smallest time unit is one full day.

Accordingly, said first period of time exceeds 8 days. More preferably,it is envisaged that said first period of time is between 8 days and 78days, with 14 or 42 days being particularly preferred.

As used herein, a time interval which is defined as “X to Y” equateswith a time interval which is defined as “between X and Y”. Both timeintervals specifically include the upper limit and also the lower limit.This means that for example a time interval “18 to 78 days” or between“18 to 78 days” includes a period of time of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,61, 62 63, 64, 65, 66, 67, 68, 69, 70 and/or 71 days.

In a more preferred embodiment of the three-stage methods/dosageregimens of the present invention, said first period of time is between3 days and 10 days, and said second period of time is between 3 days and10 days, and said third period of time is between 8 days and 78 days.

In an even more preferred embodiment, said first period of time is 7days, said second period of time is 7 days, and said third period oftime is 14 or 42 days.

In an embodiment of the three-stage methods/dosage regimens of thepresent invention, said third dose exceeds said first and second dose.Said second and third dose are preferably therapeutically active. Ofnote, said second dose exceeds said first dose.

Accordingly, in a further aspect of the three-stage methods/dosageregimens of the present invention, said first dose is between 1 and 15μg/m²/d, preferably between 5 and 15 μg/m²/d, i.e. 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 μg/m²/d. Particularly preferred is a doseof 5 or 10 μg/m²/d.

In a further aspect of the three-stage methods/dosage regimens of thepresent invention, said second dose is between 1 and 15 μg/m²/d,preferably between 5 and 15 μg/m2/d, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 μg/m²/d. Particularly preferred is a dose of 15μg/m²/d.

As used herein, a dose interval which is defined as “between X and Y”equates with a dose interval which is defined as “X to Y”. Both doseintervals specifically include the upper limit and also the lower limit.This means that for example a dose interval “between 1 and 15” or “1 to15” includes a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15μg/m²/d.

In a further aspect of the three-stage methods/dosage regimen of thepresent invention, said third dose is between 15 and 60 μg/m²/d, morepreferably between 20 and 60 μg/m²/d, i.e. 15, 20, 25, 30, 35, 40, 45,50, 55 and 60 μg/m²/d. Particularly preferred is a dose of 60 μg/m²/d.Alternatively, said third dose is between 15 and 90 μg/m²/d, morepreferably between 60 and 90 μg/m²/d, i.e., 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 70, 80 and 90 μg/m²/d.

In a preferred embodiment of the three-stage methods/dosage regimen ofthe present invention, said first dose is between 1 and 15 μg/m²/d, saidsecond dose is between 1 and 15 μg/m²/d, and said third dose is between15 and 60 or 15 and 90 μg/m²/d.

Particularly preferred, said first dose is 5 μg/m²/d, said second doseis 15 μg/m²/d, and said third dose is 60 μg/m²/d. Alternatively, saidthird dose may be 90 μg/m²/d.

As used herein, a dose interval which is defined as “between X and Y”equates with a dose interval which is defined as “X to Y”. Both doseintervals specifically include the upper limit and also the lower limit.This means that for example a dose interval “between 15 and 60” or “15to 60” includes a dose of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and/or 60μg/m²/d. Similarly, this means that for example a dose interval “between15 and 90” or “15 to 90” includes a dose of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 86, 87, 88, 89 or 90μg/m²/d.

In view of the observations made by the present inventors that athree-stage (step) method/dosage regimen aids in avoiding adverseeffects as described herein, the present invention relates to a methodof treating malignant CD19 positive lymphocytes in a human patient, saidmethod comprising (a) administering a first dose of said antibody for afirst period of time; (b) administering a second dose of said antibodyfor a second period of time; and consecutively (c) administering a thirddose of said antibody for a third period of time.

Also, the present invention relates to a method for treating malignantCD19 positive lymphocytes in a human patient, said method comprising (a)administering a first dose of said antibody for a first period of time;(b) administering a second dose of said antibody for a second period oftime; and consecutively (c) administering a third dose of said antibodyfor a third period of time.

Furthermore, the present invention relates to a method for amelioratingand/or preventing an adverse effect mediated by the administration of aCD19×CD3 bispecific antibody to a human patient, said method comprising(a) administering a first dose of said antibody for a first period oftime; (b) administering a second dose of said antibody for a secondperiod of time; and consecutively (c) administering a third dose of saidantibody for a third period of time.

Preferably, the first, second and third period of time are as describedelsewhere herein.

Regarding the doses, it is preferred that the second dose exceeds thefirst dose and the third dose exceeds the second dose as describedelsewhere herein. More preferably, the first dose is 5 μg/m²/d, thesecond dose is 15 μg/m²/d and the third dose is 60 μg/m²/d.Alternatively, the third dose may also be 90 or 120 μg/m²/d.

As noted herein above, the present invention relates to methods oftreatment/dosage regimen which employ CD19×CD3 bispecific antibodies,comprising a first binding domain capable of binding to an epitope ofhuman CD3 epsilon chain and a second binding domain capable of bindingto human CD19. Examples for bispecific molecules according to themethods of the invention are described in great detail in WO 99/54440and WO 2004/106381 and WO 2008/119565. All the specific CD19×CD3bispecific antibodies disclosed therein, including their variants,fragments, equivalents etc. are particularly preferred CD19×CD3bispecific antibodies of the present invention.

As used herein, a “CD19×CD3 bispecific antibody” (including a CD19×CD3bispecific single chain antibody) denotes a single polypeptide chaincomprising two binding domains. Such single chain antibodies arepreferred in the context of the methods/dosage regimen of the presentinvention. Each binding domain comprises at least one variable regionfrom an antibody heavy chain (“VH or H region”), wherein the VH regionof the first binding domain specifically binds to the CD3 epsilonmolecule, and the VH region of the second binding domain specificallybinds to CD19. The two binding domains are optionally linked to oneanother by a short polypeptide spacer. A non-limiting example for apolypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) (SEQ ID NO: 23)and repeats thereof. Each binding domain may additionally comprise onevariable region from an antibody light chain (“VL or L region”), the VHregion and VL region within each of the first and second binding domainsbeing linked to one another via a polypeptide linker, for example of thetype disclosed and claimed in EP 623679 B1, but in any case long enoughto allow the VH region and VL region of the first binding domain and theVH region and VL region of the second binding domain to pair with oneanother such that, together, they are able to specifically bind to therespective first and second binding domains. Such CD19CD3 bispecificsingle chain antibodies are described in great detail in WO 99/54440 andWO 2004/106381.

The term “binding domain” characterizes in connection with the presentinvention a domain of a polypeptide which specifically bindsto/interacts with a given target structure/antigen/epitope. Thus, thebinding domain is an “antigen-interaction-site”. The term“antigen-interaction-site” defines, in accordance with the presentinvention, a motif of a polypeptide, which is able to specificallyinteract with a specific antigen or a specific group of antigens, e.g.the identical antigen in different species. Said binding/interaction isalso understood to define a “specific recognition”. The term“specifically recognizing” means in accordance with this invention thatthe antibody molecule is capable of specifically interacting with and/orbinding to at least two, preferably at least three, more preferably atleast four amino acids of an antigen, e.g. the human CD3 antigen asdefined herein. Such binding may be exemplified by the specificity of a“lock-and-key-principle”. Thus, specific motifs in the amino acidsequence of the binding domain and the antigen bind to each other as aresult of their primary, secondary or tertiary structure as well as theresult of secondary modifications of said structure. The specificinteraction of the antigen-interaction-site with its specific antigenmay result as well in a simple binding of said site to the antigen.Moreover, the specific interaction of the bindingdomain/antigen-interaction-site with its specific antigen mayalternatively result in the initiation of a signal, e.g. due to theinduction of a change of the conformation of the antigen, anoligomerization of the antigen, etc. A preferred example of a bindingdomain in line with the present invention is an antibody. The bindingdomain may be a monoclonal or polyclonal antibody or derived from amonoclonal or polyclonal antibody.

The term “antibody” comprises derivatives or functional fragmentsthereof which still retain the binding specificity. Techniques for theproduction of antibodies are well known in the art and described, e.g.in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring HarborLaboratory Press, 1988 and Harlow and Lane “Using Antibodies: ALaboratory Manual” Cold Spring Harbor Laboratory Press, 1999. The term“antibody” also comprises immunoglobulins (Ig's) of different classes(i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2etc.).

The definition of the term “antibody” also includes embodiments such aschimeric, single chain and humanized antibodies, as well as antibodyfragments, like, inter alia, Fab fragments. Antibody fragments orderivatives further comprise F(ab′)2, Fv, scFv fragments or singledomain antibodies, single variable domain antibodies or immunoglobulinsingle variable domain comprising merely one variable domain, whichmight be VH or VL, that specifically bind to an antigen or epitopeindependently of other V regions or domains; see, for example, Harlowand Lane (1988) and (1999), cited above. Such immunoglobulin singlevariable domain encompasses not only an isolated antibody singlevariable domain polypeptide, but also larger polypeptides that compriseone or more monomers of an antibody single variable domain polypeptidesequence.

As used herein, CD3 epsilon denotes a molecule expressed as part of theT cell receptor and has the meaning as typically ascribed to it in theprior art. In human, it encompasses in individual or independentlycombined form all known CD3 subunits, for example CD3 epsilon, CD3delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta. The human CD3epsilon is indicated in GenBank Accession No. NM_000733.

The human CD19 protein is indicated in GenBank Accession No. AAA69966.

Preferably, the bispecific antibody applied in the methods/dosageregimens of the present invention has the domain arrangementVL(CD19)-VH(CD19)-VH(CD3)-VL(CD3).

It is, however, also envisaged that the methods of the invention can becarried out with CD19×CD3 bispecific single chain antibodies of otherdomain arrangements, such as

VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3),

VL(CD19)-VH(CD19)-VL(CD3)-VH(CD3),

VH(CD19)-VL(CD19)-VL(CD3)-VH(CD3),

VL(CD3)-VH(CD3)-VH(CD19)-VL(CD19),

VH(CD3)-VL(CD3)-VH(CD19)-VL(CD19),

VL(CD3)-VH(CD3)-VL(CD19)-VH(CD19), or

VH(CD3)-VL(CD3)-VL(CD19)-VH(CD19).

A preferred CD19×CD3 bispecific antibody applied in the methods of thepresent invention comprises the

-   -   (a) anti-CD3 CDRs of the heavy chain shown as CD3 CDR-H1 in SEQ        ID NO: 24 (RYTMH), more preferably in SEQ ID NO: 11        (GYTFTRYTMH), CD3 CDR-H2 in SEQ ID NO: 12 (YINPSRGYTNYNQKFKD)        and CD3 CDR-H3 in SEQ ID NO: 13 (YYDDHYCLDY); and/or    -   (b) anti-CD3 CDRs of the light chain shown as CD3 CDR-L1 in SEQ        ID NO: 14 (RASSSVSYMN), CD3 CDR-L2 in SEQ ID NO: 15 (DTSKVAS)        and CD3 CDR-L3 in SEQ ID NO: 16 (QQWSSNPLT); and/or    -   (c) anti-CD19 CDRs of the heavy chain shown as CD19 CDR-H1 in        SEQ ID NO: 25 (SYWMN), more preferably in SEQ ID NO: 17        (GYAFSSYWMN), CD19 CDR-H2 in SEQ ID NO: 18 (QIWPGDGDTNYNGKFKG)        and CD19 CDR-H3 in SEQ ID NO: 19 (RETTTVGRYYYAMDY); and/or    -   (d) anti-CD19 CDRs of the light chain shown as CD19 CDR-L1 in        SEQ ID NO: 20 (KASQSVDYDGDSYLN), CD19 CDR-L2 in SEQ ID NO: 21        (DASNLVS) and CD19 CDR-L3 in SEQ ID NO: 22 (QQSTEDPWT).

It is more preferred that the CD19×CD3 bispecific antibody applied inthe methods of the present invention comprises the CD3 CDRs of the heavyand light chain. Even more preferably, the CD19×CD3 bispecific antibodyapplied in the methods of the present invention comprises the CD3 CDRsof the heavy and light chain as well as the CD19 CDRs of the heavy andlight chain.

The CDRs referred to herein are in accordance with the Kabat numberingsystem. The Kabat numbering scheme is a widely adopted standard fornumbering the residues in an antibody in a consistent manner (Kabat etal., Sequences of Proteins of Immunological Interest, 1991).

Alternatively, it is preferred that the CD19×CD3 bispecific antibodyapplied in the methods of the present invention comprises the

-   (a) CD19 variable heavy chain shown in SEQ ID NO: 3 (nucleotide    sequence is shown in SEQ ID NO: 4); and/or-   (b) CD19 variable light chain shown in SEQ ID NO: 5 (nucleotide    sequence is shown in SEQ ID NO: 6); and/or-   (c) CD3 variable heavy chain shown in SEQ ID NO: 7 (nucleotide    sequence is shown in SEQ ID NO: 8); and/or-   (d) CD3 variable light chain shown in SEQ ID NO: 9 (nucleotide    sequence is shown in SEQ ID NO: 10).

More preferably, the CD19×CD3 bispecific antibody applied in the methodsof the present invention comprises the CD19 variable heavy and lightchain and/or the CD3 variable heavy and light chain. Even morepreferably, the CD19×CD3 bispecific antibody applied in the methods ofthe present invention comprises the CD19 variable heavy and light chainas well as the CD3 variable heavy and light chain.

In another alternative, it is also preferred that said bispecific singlechain antibody comprises an amino acid sequence selected from the groupconsisting of

-   (a) an amino acid sequence as depicted in SEQ ID NO: 1;-   (b) an amino acid sequence encoded by a nucleic acid sequence as    shown in SEQ ID NO: 2;-   (c) an amino acid sequence encoded by a nucleic acid sequence having    at least 70%, 80%, 90%, 95% or 99% identity to a nucleic acid    sequence of (b), wherein said amino acid sequence is capable of    specifically binding to CD3 and CD19; and-   (d) an amino acid sequence encoded by a nucleic acid sequence which    is degenerate as a result of the genetic code to a nucleotide    sequence of (b), wherein said amino acid sequence is capable of    specifically binding to CD3 and CD19.

It is to be understood that the sequence identity is determined over theentire amino acid sequence. For sequence alignments, for example, theprograms Gap or BestFit can be used (Needleman and Wunsch J. Mol. Biol.48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981),482-489), which is contained in the GCG software package (GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991). Itis a routine method for those skilled in the art to determine andidentify an amino acid sequence having e.g. 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to the amino acid sequences of theCD19×CD3 bispecific antibody described herein (preferably MT103). Forexample, according to Crick's Wobble hypothesis, the 5′ base on theanti-codon is not as spatially confined as the other two bases, andcould thus have non-standard base pairing. Put in other words: the thirdposition in a codon triplet may vary so that two triplets which differin this third position may encode the same amino acid residue. Saidhypothesis is well known to the person skilled in the art (see e.g. httpcolon-backslash-backslash en.wikipedia.org/wiki/Wobble_Hypothesis;Crick, J Mol Biol 19 (1966): 548-55). It is furthermore a routineprocedure for those skilled in the art to determine cytotoxic activityof such an amino acid sequence having e.g. 70%, 80%, 90%, 95%, 96%, 97%,98% or 99% sequence identity to the nucleotide or amino acid sequencesof the CD19×CD3 bispecific single chain antibody described herein.Cytotoxic activity of the CD19×CD3 bispecific single chain antibody oran antibody construct having e.g. 70%, 80%, 90%, 95%, 96%, 97%, 98% or99% sequence identity to the amino acid sequences of the CD19×CD3bispecific single chain antibody can be determined by methods asillustrated e.g. in WO 99/54440.

Particularly preferred, said CD19×CD3 bispecific single chain antibodyhas the amino acid sequence shown in SEQ ID NO: 1.

Also particularly preferred is the CD19×CD3 bispecific antibody MT103described in WO 99/54440 as well as those CD19×CD3 bispecific antibodiesdescribed in WO 2004/106381 and WO 2008/119565.

The present invention furthermore relates to a CD19×CD3 bispecificantibody for:

-   (i) administering a CD19×CD3 bispecific antibody to a human patient,    or-   (ii) treating malignant CD19 positive lymphocytes in a human    patient; or-   (iii) ameliorating or preventing an adverse effect mediated by the    administration of a CD19×CD3 bispecific antibody to a human patient;    wherein said antibody is to be administered in accordance with a    dosage regimen as defined in any one of the preceding claims.

Also, the present invention relates to a CD19×CD3 bispecific antibody

-   (i) administering a CD19×CD3 bispecific antibody to a human patient,    or-   (ii) treating malignant CD19 positive lymphocytes in a human    patient; or-   (iii) ameliorating or preventing an adverse effect mediated by the    administration of a CD19×CD3 bispecific antibody to a human patient;    wherein said antibody is to be administered in accordance with a    method as defined in any one of the preceding claims.

In a further aspect, the present invention concerns the use of aCD19×CD3 bispecific antibody for the preparation of a pharmaceuticalcomposition to be used in a method as defined in any one of thepreceding claims.

The pharmaceutical composition of the present invention may optionallycomprise a pharmaceutical carrier. Examples of suitable pharmaceuticalcarriers are well known in the art and include phosphate buffered salinesolutions, sterile solutions etc. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like. Furthermore, thepharmaceutical composition of the invention may comprise further agentssuch as chemotherapeutic agents as explained herein elsewhere.

In a further aspect, the present invention relates to a (pharmaceutical)kit or pharmaceutical package comprising the first dose and the seconddose as defined herein.

In another embodiment, the present invention relates to a(pharmaceutical) kit or pharmaceutical package comprising the first doseand the second dose as defined herein as well as the third dose asdefined in the context of the three-stage dosage regimen/method.

In another aspect, the (pharmaceutical) kit or pharmaceutical packagecomprises all three doses as defined herein in the context of thethree-stage dosage regimen/method, i.e., the first, the second and thethird dose.

Said first, second and third dose are thereby packaged together in onesealed pharmaceutical package or kit. It will be understood that the“first dose”, the “second dose” and the “third dose” encompasses in thisregard the respective number of single doses which will be used for agiven period of time (either the first or the second period of time).This means for example that the “first dose” or “second dose” which iscomprised in the pharmaceutical package or kit of the present inventioncomprises, for example, 7 daily doses which are separated. The number ofpackaged daily doses thereby reflects the intended period of time (Xdaily doses if said period of time is X days, Y daily doses if theperiod of time is Y days and so on). In these embodiments, the(pharmaceutical) kit or pharmaceutical package comprises the dailydosages in separate containers, in a single package.

Alternatively, it is also envisaged that the intended first dose and/orsecond dose and/or third dose is not separated into the respectivenumber of daily doses but is contained, either in toto or in part, inone single container (for example an infusion bag), which comprises therequired dose for either the first and/or the second period of timeeither in part (for example for 1 to 3 days) or in toto (i.e. for thefirst or second period of time). This means that one single containercomprises for example 7 daily doses for the “first dose” which is to beused during the first period of time etc.

It will be understood that the (pharmaceutical) kit or pharmaceuticalpackage of the present invention may also comprises more or less dailydoses as required for the respective period of time (either separated ornot). Alternatively, the (pharmaceutical) kit or pharmaceutical packageis prepared such that it contains the required number of daily doses(either separated or not) for the first and second period of time asdefined herein, i.e. the “first dose”, the “second dose” and the “thirddose” in one single package. Such a package is ideally sufficient forone complete treatment of a patient (including the first and the secondperiod of time). Parts of the kit and package of the invention can bepackaged individually in vials or bottles or in combination incontainers or multicontainer units. The manufacture of the kits followspreferably standard procedures which are known to the person skilled inthe art.

Furthermore, the invention relates to a pharmaceutical package or kit asdescribed hereinbefore and written instructions for the sequential usethereof in accordance with the methods of the present invention. Saidpharmaceutical package or kit may further comprise a label or imprintindicating that the contents can be used for treating malignant CD19positive lymphocytes present in lymphoma or leukemia in a human patient;or for ameliorating or preventing an adverse effect mediated by theadministration of a CD19×CD3 bispecific antibody to a patient.

It is also envisaged that the pharmaceutical package or kit of thepresent invention, further comprises means to administer the firstand/or the second dose and/or third dose to a patient and/or buffers,vials, teflon bags or infusion bags which are normally used for theinfusion of therapeutic agents. “Means” thereby includes one or morearticle(s) selected from the group consisting of a syringe, a hypodermicneedle, a cannula, a catheter, an infusion bag for intravenousadministration, intravenous vehicles, vials, buffers, stabilizers,written instructions which aid the skilled person in the preparation ofthe respective doses and infusions of the invention etc.

It is also envisaged that the pharmaceutical package or kit of thepresent invention further comprises a chemotherapeutic agent.

In a further aspect, the present invention provides for a pharmaceuticalpackage or kit, wherein said first and/or said second dose is arrangedsuch, that it is suitable for (prepared for) administration of a dosageregimen in accordance with a method of any one of the preceding claims.

EXAMPLES

The following examples illustrate the invention. These examples shouldnot be construed as to limit the scope of this invention. The examplesare included for purposes of illustration and the present invention islimited only by the claims.

Example 1

Identification of a Predictive Factor for Reversible NeurologicalAdverse Events in a Subset of Non-Hodgkin Lymphoma Patients Treated withCD19-Specific BiTE Antibody Blinatumomab

Blinatumomab is a CD19/CD3-bispecific antibody construct of thebispecific T cell engager (BiTE®) class showing as single agent a highrate and duration of responses in patients with relapsed non-Hodgkinlymphoma (NHL) and B-precursor acute lymphocytic leukemia (ALL).Blinatumomab has a favorable safety profile with exception of a subsetof patients developing neurological adverse events (AEs) during thefirst days of treatment, such as confusion, speech impairment orcerebellar symptoms. Thus far, all relevant neurological AEs (11 out of48 patients) were transient, fully reversible and resolved withoutsequelae within 3 to 72 hours after stop of infusion. In no case,pathological findings were seen upon cranial magnetic resonance imaging.Despite treatment discontinuation, 4 patients with neurological AEs haveachieved an objective lymphoma remission. Analysis of cerebrospinalfluid (CSF) taken within hours after stop of infusion showed detectablelevels of blinatumomab in the majority of affected patients, while inone patient without neurological symptoms no blinatumomab was detectablein CSF during infusion. Moreover, increased levels of albumin and Tlymphocytes in CSF support a disturbance of the blood brain barrier(BBB) as a possible underlying event. Analyses of patient serum samplesfor angiopoetin-2 and S1008 are ongoing to investigate whether levels ofthe endothelial stress and BBB integrity marker, respectively, correlatewith neurological AEs. In a retrospective analysis of 39 NHL patients, abaseline B cell to T cell (B:T) ratio in peripheral blood at or below1:10 was identified as the only predictive factor for the subsequentoccurrence of neurological AEs. The predictive value was thenprospectively confirmed in 8 additional patients. Of note, ALLpatients—despite very low B:T ratios—rarely showed neurological AEs,which may relate to previous intrathecal chemotherapy depleting targetcells in the brain. Potential mechanisms for the neuroprotective effectof peripheral B cells are being investigated. In conclusion, weidentified a simple measure to prospectively identify patients at riskof developing neurological AEs after onset of blinatumomab treatment.Mitigating measures are currently tested in these high-risk patients inorder to avoid discontinuation of treatment.

Example 2

Synopsis of Observations (1) in Patients Treated with a CD19×CD3Bispecific Antibody

Synopsis of Observations (1)

Common Features of Early CNS Events

-   -   First symptoms appear 12-48 h after start of MT103 infusion:        Agitation, speech impairment, sometimes tremor, ataxia    -   More severe symptoms leading to infusion stop appear after 24-72        h: Confusion, disorientation, ataxia, aphasia, seizure    -   After stop of MT103 infusion, complete resolution of CNS        symptoms seen within 1-3 days; generally no sequelae    -   Most CNS events fall into early activation and redistribution        phase of polyclonal T cells

Features of CNS Events with Slow Onset

-   -   Biased to cerebellar symptoms    -   Occur at various time points during treatment, frequently at        beginning of treatment or at step Increase    -   Tremor, mild speech impairment, mild writing impairment; can        last for several days

Other CNS Events

-   -   Additional symptoms observed without proven relationship to        other CNS events: Headache, fever, nausea

MT103 Dose Response Relationship of CNS Events

-   -   Dose response relationship of CNS events is evident; cut off is        between dose level of 5 and 15 μg/m²/d

Example 3

Synopsis of Observations (2) in Patients Treated with a CD19×CD3Bispecific Antibody

Synopsis of Observations (2)

CNS Events Appear to be Predictable

-   -   Correlation of CNS events with low B:T cell ratio (or low B cell        count)    -   B:T ratio of <1:10 identified as apparent cut off for        development of CNS events    -   No other biochemical or clinical parameters appear to correlate        with CNS events

Cranial MRI of Patients with CNS Events Mostly without PathologicalFindings CSF Analyses Suggest Opening of BBB and Neuroinfiammatory Event

-   -   Detectable levels of MT103 and increased levels of protein and        serum albumin found in majority of affected patients suggest        temporary breakdown of blood brain barrier (BBB)    -   No MT103 found in CSF of one patient free of CNS events    -   CSF analysis also shows in some affected patients increased        counts of monocytes and T lymphocytes indicative of        neuroinflammatory process    -   Are CNS events reflecting gradual opening of BBB        (agitation>confusion>aphasia, ataxia>seizure)?

Incidence of CNS Events May Correlate with Disease and/or Tumor Load

-   -   At 15 μg/m²/d, ⅜ NHL patients (37%) and only one of 1/11 ALL        ‘high risk’ patients (9%) developed CNS events    -   B-ALL patients routinely receive intrathecal chemotherapy (and        i.v. high-dose methotrexate) likely reducing tumor cell load in        CNS (“occult meningeosis neoplastica”)

Example 4

Summary of CNS Events in Patients Treated with a CD19×CD3 BispecificAntibody

Summary of Clinically Relevant CNS Events in NHL Patients First or Dosein Treatment Complete Neurological B:T cell Additional μg/m²/ Stop afterResolution, Best Patient # Assessment Disease ratio Gender, AgeTreatment Day Start Time Response 105-005 Confusion, FL 1:23.9 Female,65 First 15 15 h Yes, 24 h SD communication disorder 102-004 OrganicBrain MCL 1:757 Male, 75 First 15 50 h Yes, 34 h n.d. Syndrome 102-006Generalized MZL 1:1740 Male, 59 First 30 48 h Yes, 48 h n.d. seizure(acidosis) 109-011 Cerebellar MCL 1:9:2 Male, 73 Restart 60 48 h Yes, 24h PR Symptoms (first) 109-012 Encephalopathy MCL 1:19520 Male, 55Additional 60 24 h Yes, 24 h CR (first) 102-007 Seizure, aphasia FL1:197 Male, 61 First 90 48 h Yes, 48 h ?PR? 109-023 Encephalopathy MCL1:368 Male, 60 First 60 17 h Yes, 56 h n.d. 109-025 Encephalopathy MCL1:873 Male, 58 First 15 41 h Yes, 48 h n.d. 108-004 Speech FL 0:431Male, 66 First 60 624 h  Yes, 3 h PR Impairment Palsy Face and Arm109-261 Desorientation, MCL 1:20 Male, 42 Additional 60 30 h Yes, 72 hPR Speech (first Impairment cycle)

Example 5

Dose Dependency of CNS Events of Patients Treated with a CD19×CD3Bispecific Antibody in Clinical Trials

Dose Dependency of CNS Events in Ongoing NHL Trial ‘High risk’ patientsdefined by having low B:T cell ratio (<1:10) Initial dose considered forclassification in dose groups Dose All ‘High Risk’ ‘Low Risk' ≤5  0/14(0%)  0/4+ (0%)     0/10 (0%)  15 3/16 (19%) 3/8+ (38%)   0/8 (0%) 30 1/6 (17%) 1/1 (100%) 0/5 (0%) 60 5/13 (38%) 4/5 (80%)  1*/8 (13%) 90 2/3 (66%) 1/1 (100%)  1/2 (50%) All 11/52^(§) (21%)  9/19 (47%)  2/33(6%)  ^(§)>48 patients is due to additional treatments and re-starts ofindividual patients (resulting in conversion to ‘high risk’) *Reachedborderline B:T ratio after first treatment cycle +Incl. patients withstep-wise dose increase

Example 6

A Patient Having an Increased Risk of Potential Adverse Effects WhoReceived 15 μg/m²/d for 7 Days and 60 μg/m²/d for 21 Days Showed NoAdverse Effects (Neurological Reactions)

Patient 108-003

-   -   Female, 66 y    -   FL grade 2, IVB (FD: September 2006)    -   Relevant medical history: anemia, thrombocytopenia,        (pre-treatment 2× Zevalin and bone marrow infiltration by FL)        elevation of gGT and AP, abuse of benzodiazepines, status        after 2. aureaus sepsis with spondylodiscitis and abscesses    -   Prior lymphoma treatment:        -   6× R-CHOP 14, 8× R September 2006-February 2007        -   R mono May 2007        -   1. Zevalin November 2007        -   2. Zevalin January 2008

Patient 108-003

-   -   According to initial B:T cell ratio (1:10.5) high-risk (cohort        15/60)    -   Jan. 5, 2009 Treatment start (15 μg/m²/24 h)    -   Fever, headache for 2 days—easily handled by oral paracetamol        and novalgin    -   January 12th dose increase to 60 μg/m²/24 h    -   Again fever, headache—easily handled by oral paracetamol and        novalgin    -   No neurological events    -   Well tolerated dose “step”    -   Suspected improvement of bone marrow function

Example 7

A Patient Having an Increased Risk of Potential Adverse Effects WhoReceived 5 μg/m²/d for 7 Days and 60 μg/m²/d for 21 Days Showed MildAdverse Effects (Neurological Reactions)

-   -   MCL, male 42 y    -   B:T 1:12    -   Treatment start Jan. 19, 2009 with 5 micg/m2/d    -   Day 1: fever and chills, headache, no further problems    -   Step: January 26th: after 6 h fever, strong headache    -   27, Jan. 2009: tiredness, nausea, vomiting, endoscopy without        pathological findings), absolute arrhythmia with frequency up to        170/min→resolution within one day after substitution of        potassium and digitoxin.    -   Cranial CT scan and CSF perfomed, CT: no pathological findings    -   CSF: slightly elevated protein 55 mg/dL, cells: 23 Zellen/micL,        mainly monocytic cells and some activated lymphocytes    -   27, Jan. 2009 afternoon: mild tremor, apraxia, “slow mental        state”, evening: mild speech impairment (cerebellar?), slow        improvement over the next two days 29, Jan. 2009 due to ongoing        mild symptoms decision to give dexamethasone    -   Slow improvement of symptoms, complete resolution 31, Mar. 2009    -   During the further course of treatment: recurrent difficulties        to play the guitar.    -   After 4 weeks treatment: −37%    -   After 8 weeks of treatment: PR/CRu

Example 8

A Patient Having an Increased Risk of Potential Adverse Effects WhoReceived a Treatment Regimen According to the Present Invention.

Patient 108-005

-   -   Male, 71 y, FL IIIB    -   13:T cell ratio: 57:1363 (low, 1:23.9)    -   First diagnosis: 1997    -   Multiple prior treatments: 12× Rituximab (mono), 6×        Rituximab-Bendamustin, 6× R-CHOP, autologous SCT    -   Date of Blinatumomab start: 17.8.2009    -   Treatment duration: 8 weeks    -   Well tolerated (no SAE)    -   No neurological adverse event    -   8 Week CT Scan: −65%=partial remission of the lymphoma

Example 9

A Further Patient Having an Increased Risk of Potential Adverse EffectsWho Received a Treatment Regimen According to the Present Invention.

Patient 109-031

-   -   Male, 60 y, Follicular Lymphoma IVAE    -   B:T cell ratio: 0:429 (low)    -   First diagnosis: May 2009    -   Prior treatments: Pre-phase w. Vincristin/Decortin, 6× R-CHOP    -   Blinatumomab treatment Start: 30, Nov. 2009    -   Treatment duration: 8 weeks    -   Well tolerated (flush symptoms at steps—responsive to steroids)    -   No neurological adverse event    -   Lymphoma −56% after 8 weeks (partial remission of the lymphoma)

The invention claimed is:
 1. A method for treating B-lineage acutelymphoblastic leukemia (ALL) in a human patient, said method comprising:(a) determining the total B cell number and total T cell number in aperipheral blood sample of said patient; (b) calculating the B:T cellratio from the total B cell number and the total T cell number in aperipheral blood sample of said patient; and (c) administering to thepatient having a B:T cell ratio of 1:5 or lower a CD19×CD3 bispecificsingle chain antibody comprising two binding domains: (i) a CD19 bindingdomain comprising anti-CD19 heavy chain CDRs of CDR-H1 as set forth inSEQ ID NO: 17, CDR-H2 as set forth in SEQ ID NO: 18, and CDR-H3 as setforth in SEQ ID NO: 19, and anti-CD19 light chain CDRs of CDR-L1 as setforth in SEQ ID NO: 20, CDR-L2 as set forth in SEQ ID NO: 21 and CDR-L3as set forth in SEQ ID NO: 22; and (ii) a CD3 binding domain comprisinganti-CD3 heavy chain CDRs of CDR-H1 as set forth in SEQ ID NO: 11,CDR-H2 as set forth in SEQ ID NO: 12, and CDR-H3 as set forth in SEQ IDNO: 13; and anti-CD3 light chain CDRs of CDR-L1 as set forth in SEQ IDNO: 14, CDR-L2 as set forth in SEQ ID NO: 15, and CDR-L3 as set forth inSEQ ID NO: 16 in a step dose regimen, wherein in the step dose regimen afirst dose of between 5 and 15 μg/m²/d is administered for a firstperiod of time of at least three days, a second dose of between 5 and 15μg/m²/d of said CD19×CD3 bispecific antibody is subsequentlyadministered for a second period of time, wherein the second period oftime exceeds the first period of time, and wherein the second doseexceeds the first dose, and a third dose of between 15 and 60 μg/m²/d isadministered for a third period of time.
 2. The method of claim 1,wherein the route of administration is intravenous.
 3. The method ofclaim 1, wherein said second period of time exceeds 18 days.
 4. Themethod of claim 3, wherein said second period of time is between 18 daysand 81 days.
 5. The method of claim 1, wherein said second period oftime exceeds 3 days.
 6. The method of claim 1, wherein said secondperiod of time is between 3 days and 10 days.
 7. The method of claim 1,wherein said third period of time exceeds 8 days.
 8. The method of claim1, wherein said third period of time is between 8 days and 78 days. 9.The method of claim 1, wherein said antibody is administered at a firstdose of 5 μg/m²/d, followed by a second dose of 15 μg/m²/d andconsecutively followed by a third dose of 60 μg/m²/d.
 10. The method ofclaim 1, wherein the CD19×CD3 bispecific antibody comprises (a) theanti-CD19 variable heavy chain as set forth in SEQ ID NO: 3 and theanti-CD19 variable light chain as set forth in SEQ ID NO: 5; and (b) theanti-CD3 variable heavy chain as set forth in SEQ ID NO: 7 and theanti-CD3 variable light chain as set forth in SEQ ID NO:
 9. 11. Themethod of claim 1, wherein the CD19×CD3 bispecific antibody comprises anamino acid sequence comprising at least 70% identity to the amino acidsequence as set forth in SEQ ID NO:
 1. 12. The method of claim 11,wherein the CD19×CD3 bispecific antibody comprises the amino acidsequence as set forth in SEQ ID NO:
 1. 13. The method of claim 1,wherein the CD19×CD3 bispecific antibody comprises an amino acidsequence comprising at least 80% identity to the amino acid sequence asset forth in SEQ ID NO:
 1. 14. The method of claim 1, wherein theCD19×CD3 bispecific antibody comprises an amino acid sequence comprisingat least 90% identity to the amino acid sequence as set forth in SEQ IDNO:
 1. 15. The method of claim 1, wherein the CD19×CD3 bispecificantibody comprises an amino acid sequence comprising at least 95%identity to the amino acid sequence as set forth in SEQ ID NO:
 1. 16.The method of claim 1, wherein the CD19×CD3 bispecific antibodycomprises an amino acid sequence encoded by a nucleotide sequencecomprising at least 70% identity to the nucleotide sequence as set forthin SEQ ID NO:
 2. 17. The method of claim 1, wherein the CD19×CD3bispecific antibody comprises an amino acid sequence encoded by anucleotide sequence comprising at least 80% identity to the nucleotidesequence as set forth in SEQ ID NO:
 2. 18. The method of claim 1,wherein the CD19×CD3 bispecific antibody comprises an amino acidsequence encoded by a nucleotide sequence comprising at least 90%identity to the nucleotide sequence as set forth in SEQ ID NO:
 2. 19.The method of claim 1, wherein the CD19×CD3 bispecific antibodycomprises an amino acid sequence encoded by a nucleotide sequencecomprising at least 95% identity to the nucleotide sequence as set forthin SEQ ID NO:
 2. 20. The method of claim 1, wherein the CD19×CD3bispecific antibody comprises an amino acid sequence encoded by thenucleotide sequence as set forth in SEQ ID NO: 2.